• Purity

  >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to phosphorylate 1-deoxy-1-morpholino-D-fructose. The specific activity is >30 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Glu2-Lys309, with N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  36 kDa

• SDS-PAGE

  37 kDa, reducing conditions

Assay Procedure

Materials

• Universal Kinase Activity Kit (Catalog # )

• 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0

• Recombinant Human Fructosamine-3-kinase/FN3K (rhFN3K) (Catalog # 9155-FU)
   Substrate: 1-Deoxy-1-morpholino-D-fructose (Sigma, Catalog # D6149), 40 mM stock in deionized water

• 96-well Clear Plate (Catalog # )

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.

2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity kit by adding 40 μL of the 1 mM Phosphate Standard to 360 μL of 1X Assay Buffer for a 100 μM stock. This is the first point of the standard curve.

3. Complete the standard curve by performing six one-half serial dilutions of the 100 μM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.

4. Prepare a reaction mixture containing 0.4 mM ATP (supplied in kit) and 2 mM 1-Deoxy-1-morpholino-D-fructose in 1X Assay Buffer.

5. Dilute rhFN3K to 33.3 μg/mL in 1X Assay Buffer.

6. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.

7. Load 15 μL of 33.3 μg/mL rhFN3K into empty wells of the same plate as the curve. Include a control containing 15 μL of 1X Assay Buffer.

8. Add 25 μL of reaction mixture to the wells, excluding the standard curve.

9. Seal plate and incubate at 37 °C for 30 minutes.

10. Dilute Coupling Phosphatase 4 (supplied in kit) to10 μg/mL in 1X Assay Buffer.

11. Add 10 μL of 10 μg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.

12. Seal plate and incubate at room temperature for 5 minutes.

13. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.

14. Add 100 μL of deionized water to all wells. Mix briefly.

15. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

16. Read plate at 620 nm (absorbance) in endpoint mode.

17. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.
     **Decoupled reaction (use incubation time of 35 minutes).

Per Reaction:

• rhFN3K: 0.5 μg

• Coupling Phosphatase 4: 0.1 μg

• ATP: 0.2 mM

• 1-Deoxy-1-morpholino-D-fructose: 1 mM

Background: Fructosamine-3-kinase/FN3K

Nonenzymatic glycation (Maillard reaction) is a process in which glucose and other sugars react spontaneously with amine-containing molecules, such as proteins (1). These reactions proceed through many stages, starting with glucosylamines (Schiff bases), fructosamines (Amadori compounds) (2), and aminoaldoses (Heyns compounds) (3) and ultimately lead to the formation of irreversible end products, which include crosslinks, aromatic heterocycles, and oxidized compounds (4), that are designated collectively as advanced glycation end products (AGEs) (5). The concentrations of these products are elevated in diabetes, and important in the etiology of diabetes complications (6, 7). Fructosamine-3-phosphokinase (FN3K) is a kinase that phosphorylates fructoselysine and a wide variety of fructosamines, therefore removing these AGEs (8-10). Lost the protective enzymatic activity of FN3K is also associated with the oncology of colorectal cancer (11). FN3K is particularly active in brain, heart, kidney, and skeletal muscle and in erythrocytes (12,13). The kinase activity of recombinant human PFKFB3 was assayed using a phosphatase-coupled method (14).

• References:

1. Monnier, V.M. (1989) Prog. Clin. Biol. Res. 304:1.

2. Roper, H, et al. (1994). Carbohydr. Res. 262:257.

3. Carson, J.F. et al. (1955). J. Am. Chem. So.c 77:1881.

4. Reddy, S, et al. (1995) Biochemistry 34:10872.

5. Bucala, R. and Cerami, A. (1992) Adv. Pharmacol. 23:1.

6. Lyons, T.J. and Jenkins, A.J: (1997). Diabetes Rev. 5:365.

7. Furusyo, N and Hayashi, J. (2013) Biochim. Biophys. Acta. 1830:5509.

8. Lal, S, et al.(1993) J. Biol. Chem. 268:7763.

9. Szwergold, B.S. et al. (2001) DIABETES 50:2139.

10. Delpierre, G. et al. (2002) Biochem. J. 365: 801.

11. Caruso, M.G. et al (2007) Oncology 73(1-2):72.

12. Delplanque, J. et al. (2004) J. Biol.Chem. 279:46606.

13. Petersen A, et al. (1992) Biochem J 284:363.

14. Wu, Z.L. et al. (2011) PloS ONE 6(8):e23172.

• Entrez Gene IDs:

  64122 (Human); 63828 (Mouse)

• Alternate Names:

  EC 2.7.1.-; FN3K; fructosamine 3 kinase; Fructosamine-3-kinase